1. Field of the Invention
The present invention concerns novel Pseudorabies virus recombinants and the use of such recombinants in the production of desired proteins.
2. State of the Art
Developments in genetic engineering have made it possible to isolate DNA fragments containing specific genes or portions thereof from the genome of one organism or microorganism and insert those fragments into another DNA molecule called a vector. The recombinant construct thus created is then introduced into a host such as a bacterial, yeast, insect or animal cell, where it is propagated and, if desired, the protein encoded by the fragment insert produced. The genetic information required for the recombinant construct's propagation in the host is generally provided by the vector molecule.
Because of problems inherent in the use of large scale animal and insect cell cultures, much of the effort in this area has been directed to utilizing bacteria and yeasts as host organisms. However recent advances in cell culture techniques have made the use of large scale cultures a feasible means to produce commercially important proteins. Animal and insect cells offer sophisticated protein processing capabilities such as glycosylation, methylation, phosphorylation and sugar and lipid association which are often unavailable in bacterial or yeast systems. Since many proteins products must be glycosylated or otherwise processed for activity, recombinants capable of propagation and expression in such hosts yield enormous promise for the production of a variety of enzymes, hormones, antigens, antibodies and other proteins having medical, agricultural or industrial utility. Viruses, with their ability to infect, replicate and express proteins in animal and insect cells as well as other hosts, provide a starting point for the creation of recombinants directed to these ends. Viruses engineered to contain foreign genes coding for antigens could also be employed as live vaccines.
European patent application 0,073,656 dated Aug. 26, 1982 discusses, inter alia, the production of hepatitis surface antigen in vertebrate cell cultures tranformed with recombinant DNA constructed using a simian virus 40 (SV40) with an excised VP-1 gene, E. coli plasmid pBR322, and the gene encoding the surface antigen of hepatitis B virus, HBsAg. SV40 is a small DNA virus infecting some strains of monkeys. It is not known to be pathogenic for humans but has been found to cause tumors in animals.
The construction of hybrid vaccinia viruses by genetic engineering to produce live vaccines is described by Smith et al., Proc. Natl. Acad. Sci. USA, 80:7155-7159, (1983). The authors discuss the insertion and expression of the influenza virus hemagglutinin gene in vaccinia virus, a virus with a large DNA genome. The foreign gene was inserted into the thymidine kinase gene because that gene is nonessential for viral replication and simultaneously provided a selective process for recombinant viruses. Cells infected with the engineered vaccinia virus produced glycosylated hemagglutinins and the virus successfully immunized hamsters against infection with influenza virus. The vaccinia virus can be grown in the cells of numerous animal and avian species. However, a general concern with vaccinia virus is that it is infectious for humans and has been known to cause disease.
Roizman et al., Science 229: 1208-1214 (1985) points out that one of the central objectives of studies on the molecular biology of viruses infecting humans and animals is the specific modification of the viral genomes for use as vaccines or as vectors of genes whose products provide protection against infectious disease. They describe strategies to engineer another large DNA virus, herpes simplex virus type 1 (HSV-1), a human pathogen, and note the problems inherent in using large DNA viruses as vectors for foreign DNA. Shih et al., Proc. Natl. Acad. Sci. USA, 81: 5867-5870, (1984), describe a HSV-1 engineered to cause infected cells to produce and excrete the Hepatitis B virus surface antigen. HSV-1 is a common human pathogen that establishes itself in the latent state often resulting in a recurring infection--cold sores. The latent state and recurrence of infections persists for the rest of the life of the infected person.
While it has been established that the DNA of SV40, HSV-1, vaccinia and a few others can be used as vectors for the introduction and expression of foreign genes in cellular environments, there is no question that the development of other viral vectors having similar capabilities but lacking the liabilities of many of those currently employed would be of great importance to the medical, agricultural and industrial community.
U.S. Pat. No. 4,514,497 discloses the use of the pseudorabies virus as a vaccine for pigs. Pseudorabies is a large DNA virus and is a known pathogen for swine, causing the syndrome known as Aujusky's disease. To be used as a vaccine, the virus was attenuated by excising or altering its thymidine kinase gene. No additional genes were added to the virus and it vaccinated the pigs only against pseudorabies.